Preparation of monovalent follistatin-like 3-Fc-fusion protein and evaluation of its effects on muscle mass in mice

Figures

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  Figure 1. Schematic presentation of monovalent FSTL3-Fc protein The mono-FSTL3-Fc heterodimer is generated through co-expression of FSTL3-Fc(T366W, knob) and Fc(T366S/L368A/Y407V, hole) constructs which harbor the knobs-into-holes mutations.

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  Figure 2. SDS-PAGE of purified recombinant mono-FSTL3-Fc (A) Schematic presentation of mono-FSTL3-Fc in either non-reduced or reduced conditions. The predicted protein molecular weight (MW) was calculated by the sum of the MW of all amino acids. (B) Results of Coomassie Brilliant Blue (CBB) staining and immunoblot analysis for human IgG Fc. It is of note that FSTL3 is a target of post-translational modification including glycosylation, which may affect the molecular weights.

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  Figure 3. Representative macroscopic images of the mouse hindlimb muscles (A) Ventral view of the hindlimb region. (B) Isolated skeletal muscles. TA: tibialis anterior, Ham: hamstrings, QF: quadriceps femoris, GC: gastrocnemius. Scale: cm.

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  Figure 4. Schematic presentation of skeletal muscle sample preparation for cryosectioning (A) Cover the muscle in a small amount of fresh embedding medium such as FSC 22 frozen section medium, and cut the muscle at the mid-belly region. (B and C) Place the sample with the cut surface down on the aluminum foil, and dip the aluminum foil with the muscle into the isopentane/acetone bath for 10–20 s (D and E) Embed the frozen muscle into FSC 22 frozen section medium in a tissue-embedding mold, and dip the mold with the muscle into the isopentane/acetone bath.

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  Figure 5. Quantification of the muscle fiber cross-sectional area (CSA) (A and B) Representative cross-sectional images of myofibers in the GC muscle excised from mice with 2-week intraperitoneal administration of mono-FSTL3-Fc. A: immunohistochemistry (IHC) for laminin (red) with DAPI nuclear stain (blue), B: quantification using Hybrid Cell Count software. Scale bar: 100 μm.